Protein for use in hypoxia related conditions

ABSTRACT

The present invention relates to new uses of Solute Carrier 6 (SC6), a taurine transporter, in the diagnosis, screening, treatment and prophylaxis of hypoxia related conditions e.g. cancer. Compositions comprising the protein, such as vaccines and agents that modulate the protein expression or activity, including antibodies that are immunospecific for the protein, are also provided.

The present invention relates to new uses of Solute Carrier 6 (SC6), ataurine transporter, in the diagnosis, screening, treatment andprophylaxis of hypoxia related conditions e.g. cancer. Compositionscomprising the protein, such as vaccines and agents that modulate theprotein expression or activity, including antibodies that areimmunospecific for the protein, are also provided.

Solute Carrier 6

The human solute carrier 6 gene, NCBI accesion no. NM_(—)003043, wascloned and characterised by Ramamoorthy S, et al., (1994, Biochem J, 300(Pt 3), 893-900) and encodes a taurine transporter protein. The cDNA wasisolated from a human placental cDNA library and is highly related tothe rat brain taurine transporter (U.S. Pat. No. 5,658,786).Transfection of this cDNA into HeLa cells results in a marked elevationof taurine transport activity. The activity of the cDNA-inducedtransporter is dependent on the presence of Na⁺ as well as Cl⁻. TheNa⁺/Cl⁻/taurine stoichiometry for the cloned transporter is 2:1:1. Thetransporter is specific for taurine and other beta-amino acids,including beta-alanine, and exhibits high affinity for taurine(Michaelis-Menten constant approximately 6 microM). The nucleotidesequence of the coding region predicts a 620-amino acid protein with acalculated M(r) of 69,853 Da (Ramamoorthy S, et al., 1994).

Cellular Function of Taurine

Taurine (2-aminoethane sulphonic acid) a ubiquitous beta-amino acid isconditionally essential in man. It is derived from methionine andcysteine and is not utilized in protein synthesis but found free or insome simple peptides. Intracellular taurine is generally maintained athigh concentrations although its' role appears to be cell-specific.Plasma taurine levels are also high, although decreases have beenobserved in response to surgical injury and numerous pathologicalconditions including cancer and sepsis (Stapleton P P, et al., (1998) JParenter Enteral Nutr, January-February, 22(1), 42-8).

Taurine is a known neuromodulator and is an inhibitory amino acid.Another key function is as an osmolyte in most cells. Thus, it mayregulate many biological processes, including heart rhythm, contractilefunction, blood pressure, platelet aggregation, neuronal excitability,body temperature, learning, motor behavior, food consumption, eye sight,sperm motility, cell proliferation and viability, energy metabolism andbile acid synthesis. Many of these actions are associated withalterations in either ion transport or protein phosphorylation. Althoughthe effects on ion transport have been attributed to changes in membranestructure, they could be equally affected by a change in the activity ofthe affected transporters. The transporter activity can be altered byenhanced protein expression, changes in the phosphorylation status ofthe protein and cytoskeletal changes. Interestingly, all three eventsare altered by osmotic stress (Schaffer S, et al., (2000) Amino Acids,19(34), 52746).

Additionally, taurine has been associated with hypoxia, hypoglycemia,ischemia, antioxidation (oxidative stress and the prescence of freeradicals) detoxification, and stimulation of glycolysis andglycogenesis.

A potentially important taurine role is in cell protection againsthypoxia (Canas P E (1992) Acta Physiol Pharmacol Ther Latinoam, 42(3),133-7). Studies on renal cell cultures have shown that when taurine wasadministered during hypoxia, the concomittant cell damage was markedlyreduced. Hence, it reduced the osmoregulatory deterioration duringhypoxia and reoxygenation, so that calcium homeostasis was markedlyimproved. Furthermore, Ca²⁺ efflux during hypoxia as well as Ca²⁺overload during reoxygenation was significantly reduced. The effect oftaurine was partly comparable to the effect induced by Ca²⁺ channelblockers. One of the effects mainly responsible for cellular protectionappears to be the taurine-induced acceleration of cellular growthprocesses in spite of hypoxia and reoxygenation. The spectrum ofcytoprotective effects of taurine predisposes this substance to be aphysiological protective agent responsible for cellular homeostasis orenantiostasis (Michalk D V et al., (1996) Adv Exp Med Biol, 403,223-32). In neuron damaging conditions it may constitute an importantprotective mechanism against excitotoxicity, e.g. ischemia (SaransaariP, Oja S S, (2000) Amino Acids 19 (34), 509-26).

Lastly, taurine, arginine and homocysteine are amino acids which havebeen shown to affect the risk factors of cardiovascular diseases inhumans (Nittynen L, et al., (1999) Ann Med, October; 31(5), 318-26).

Hypoxia and Tumour Growth

Tumour growth is dependant on oxygen and nutrients supplied by the localtissue vasculature. Solid tumours are well known to be poorly oxygenatedcompared to normal tissue (In: Vaupel, P. W. et al., (eds.) TumourOxygenation pp219-232: Gustav Fisher Verlag, 1995). Hypoxia (lowcellular oxygen concentration, <1%) arises when tumour cells proliferateoutside the diffusion zone of the local vascular supply. Tumours respondto hypoxia by producing hypoxia inducible factors (e.g. VEGF) thatstimulate the growth of endothelial cells (the cells lining bloodcapillaries) from surrounding blood vessels (i.e. angiogenesis) (WeidnerN, et al., N Engl J Med, 1991, Jan. 3, 324:1, 1-8). Blood flow in thesetumour blood vessels is sluggish and irregular which results in lessefficient oxygen delivery and propagates the hypoxic tendency of tumours(for review see, Brown J. M. Mol Med Today 6; 157-62, 2000). Thishypoxia-induced angiogenic process allows turnout cells access to thehost animal's circulatory system. Furthermore, the new blood vesselsprovide a gateway for tumor cells to enter the circulation andmetastasize to distant sites (Folkman J, J Natl Cancer Inst, 1990, Jan.3; 82(1):4-6). In fact, the extent of neovascularity is stronglycorrelated with metastases in primary breast carcinoma, bladder cancer,prostate cancer, non-small cell lung cancer, cutaneous melanomas anduterine cervix carcinoma (reviewed in: Ferrara N, Breast Cancer ResTreat, 1995, 36:2, 127-37). These findings have led researchers tospeculate that tumor vascularisation could be used as a diagnostic toolto predict the stage of the cancer, i.e. whether the cancer hasmetastasized or not and to what extent.

Hypoxia in Tumour Therapy

Carcinomas are known to have significant hypoxic fractions, e.g. 80% ofthe tumour for head and neck squamous cell carcinomas and 50% of thetumour for carcinoma of the uterine cervix (Van De Wiele, C et al.,(2001) Nuclear Med, 22, 945-947). The hypoxic areas are heterogeneousand are partly due to the different oxygen tensions present throughoutthe tumour. Hypoxic areas of turnouts tend to escape radiation andchemotherapy. These areas are the furthest away from blood vessels andhence receive poor drug delivery. Hypoxic tumour cells also tend to beslow-proliferating and most chemotherapy drugs target rapidly dividingtumour cells only. This may be one of the reasons why hypoxia can inducerelapse after treatment and the evolution of more aggressive andresistant tumours. Hypoxia increases the mutation rate of cells andresults in mutated cell-types that are less susceptible to programmedcell death signals, such as p53. Overall, turnout hypoxia has emerged asa predictor of poor prognosis. Since hypoxia is an abnormal conditionthat exists in all solid tumours, hypoxic cells could be used as a veryspecific target for anti-cancer therapies.

Angiogenesis

Under normal conditions, angiogenesis is necessary to facilitate woundhealing, tissue repair, reproduction, growth and development. However,many disease states are also dependent upon this process. The process ofwound healing is complex and represents a serious medical problemaffecting a large number of individuals. Healing problems occur indermal wounds, such as decubitus ulcers, severe burns, diabetic ulcersand eye lesions (including dry eye and corneal ulcers) as well assurgical wounds and other wound-related pathologies. One importantaspect of wound healing is the controlled migration of new cells fromtissues surrounding the wound-site. This is in order to establish aproper population of cell types and correct tissue organization in thenewly developing tissue. Hypoxia promotes increased vascular growth andis thus associated with tumour growth as described above. Additionally,excessive vascular growth is also known to contribute to non-neoplasticdisorders, such as diabetic retinopathy, asthma, macular degeneration,psoriasis and rheumatoid arthritis.

Therefore, a need exists to identify new markers and potential targetsof the oxygen-regulated angiogenic pathway that are important in thedevelopment of cancers and that can be used for the diagnosis andtreatment of cancer.

An ideal protein target for cancer immunotherapy should have arestricted expression profile in normal tissues and be over-expressed intumours, such that the immune response will be targeted to tumour cellsand not against other organs. In addition, the protein target should beexposed on the cell surface, where it will be accessible to therapeuticagents. Tumour antigens have been identified for a number of cancertypes, by using techniques such as differential screening of cDNA(Hubert, R. S., et al., Proc. Natl. Acad, Sci. USA 96, 14523-14528(1999); Lucas, S., De Plaen, E. & Boon, T. Int. J. Cancer 87, 55-60(2000)), and the purification of cell-surface antigens that arerecognised by tumour-specific antibodies (Catimel, B., et al., J. Biol.Chem. 271, 25664-25670 (1996)).

The present invention is based on the finding that SC6 is upregulated inthe cellular response to hypoxia. Our findings indicate that SC6expression is increased under hypoxic conditions in human dermalmicrovascular endothelial (HDMEC) and renal cancer (RCC4) cell lines,indicating that it responds to a hypoxic stimulus. In addition, SC6 wasexpressed in relatively low amounts in many normal tissues investigated.However, SC6 expression was elevated in some clinical tissue samples ofcervical, colon, renal, lung and uterine cell carcinomas, in clinicallymphoma samples and in certain tumour cell lines, e.g. Burkitt'slymphoma, myeloid and T-cell leukaemia, breast, renal and pancreaticcarcinoma cell lines, indicating that SC6 plays a role in thedevelopment of many cancer types and may be a suitable target for cancertherapy and diagnosis.

The amino acid sequence for SC6, can be found in the GenBank database,held by the National Institute of Health (NIH) available athttp://www.ncbi.nlm.nih.gov/), under accession number NP_(—)003034 (SEQID No. 1) and the nucleic acid sequence is shown under accession numberNM_(—)003043 (SEQ ID No. 2).

The present invention contemplates the use of an SC6 protein e.g.full-length, in the diagnosis, prognosis and treatment of hypoxiarelated conditions, in particular cancer, such as but not limited to,cervical, colon, renal, lung, uterine, breast or pancreatic cellcarcinoma, lymphoma e.g. Burkitt's lymphoma, leukaemia e.g. myeloid orT-cell leukaemia, angiogenesis and angiogenesis related disorders, suchas, diabetic retinopathy, asthma, macular degeneration, psoriasis andrheumatoid arthritis. The use of therapeutic antibodies raised againstthe extracellular portion of SC6 is specifically contemplated.

Accordingly, the present invention provides a method of screening forand/or diagnosis of hypoxia related conditions in a subject and ormonitoring the effectiveness of therapy for said condition, whichcomprises the step of detecting and/or quantifying in a biologicalsample obtained from said subject an SC6 polypeptide which

-   -   a) comprises or consists of the amino acid sequence of SEQ ID        No. 1;    -   b) is a variant having one or more amino acid substitutions,        deletions, insertions or modifications relative to the amino        acid sequence of SEQ ID No. 1, provided that such variant        exhibits the immunological and/or transporter activity of the        polypeptide with the amino acid sequence of SEQ ID No. 1; or    -   c) is a fragment of a polypeptide as defined in a) or b) above,        which is at least ten amino acids long.

In one embodiment, the amount of the SC6 polypeptide is compared to areference range or control. Preferably, the hypoxia related condition iscancer, e.g. hypoxic cancerous cells. A convenient means for suchdetection/quantifying will involve the use of antibodies. Accordingly,in one embodiment of the method described above the polypeptide isdetected and/or quantified using an antibody that specifically binds toone or more SC6 polypeptides.

The term “SC6 polypeptide” hereinafter includes polypeptides asdescribed in a) to c) above.

The term “hypoxia related condition” includes cancer, such as but notlimited to, cervical, colon, renal, lung, uterine, breast or pancreaticcell carcinoma, lymphoma e.g. Burkitt's lymphoma, leukaemia e.g. myeloidor T-cell leukaemia, angiogenesis and angiogenesis related disorders,such as but not limited to, diabetic retinopathy, asthma, maculardegeneration, psoriasis and rheumatoid arthritis. “Cancer” encompassesboth leukaemia and lymphoma. “Leukaemia” is the term used to describe anacute or chronic disease that involves the blood forming organs and ischaracterised by an abnormal increase in the number of leucocytes in thetissues of the body with or without a corresponding increase of those inthe circulating blood and is classified according of the type ofleucocyte most prominently involved. “Lymphoma” is the term used todescribe a malignant tumour of lymphoblasts derived from B lymphocytes.

The term “biological sample” includes fluid, e.g. serum or lymph, andtissue, e.g. breast tissue, samples.

The term “agent” refers to, without limitation, agents which interactwith and/or modulate the expression or activity of an SC6 polypeptidee.g. antibodies that bind to an SC6 polypeptide, agonists or antagonistsof an SC6 polypeptide and molecules, e.g. chemical compounds, orbio-molecules, e.g. peptides.

The present invention also provides a method of screening for and/ordiagnosis of hypoxia related conditions in a subject and/or monitoringthe effectiveness of therapy for said condition, which comprises thestep of detecting and/or quantifying in a biological sample obtainedfrom said subject the amount of a DNA nucleic acid sequence which,

-   -   a) comprises or consists of the DNA sequence of SEQ ID No. 2, or        its RNA equivalent;    -   b) is a sequence which is complementary to the sequences of a);    -   c) is a sequence which codes for the same polypeptide, as the        sequences of a) or b);    -   d) is a sequence which shows substantial identity with any of        those of a), b) and c); or    -   e) is a sequence which codes for a variant or fragment of the        polypeptide with the amino acid sequence of SEQ ID No. 1.

It is preferred if sequences which show substantial identity with any ofthose of a), b) and c) have e.g. at least 50%, at least 75%, at least80%, at least 85%, at least 90%, at least 95% or at least 98% sequenceidentity.

The term “SC6 nucleic acid molecules” hereinafter includes nucleic acidsequences as described in a) to e) above.

In a further aspect, the method of detecting the presence of an SC6polypeptide comprises detecting the captured polypeptide using adirectly or indirectly labelled detection reagent.

An SC6 polypeptide can be detected by a number of methods known to thoseskilled in the art. The methods of diagnosis according to the presentinvention may be performed using the following methods known to thoseskilled in the art, including, without limitation, immunoprecipitationfollowed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, 2dimensional gel electrophoresis, competitive and non-competitive assaysystems using techniques such as Western blots, immunocytochemistry,immunohistochemistry, immunoassays, e.g. radioimmunoassays, ELISA(enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays and protein A immunoassays.

The invention also provides diagnostic kits, comprising a capturereagent (e.g. an antibody) against an SC6 polypeptide. In addition, sucha kit may optionally comprise one or more of the following:

-   (1) instructions for using the capture reagent for diagnosis,    prognosis, therapeutic monitoring or any combination of these    applications;-   (2) a labelled binding partner to the capture reagent;-   (3) a solid phase (such as a reagent strip) upon which the capture    reagent is immobilised; and-   (4) a label or insert indicating regulatory approval for diagnostic,    prognostic or therapeutic use or any combination thereof.

If no labelled binding partner to the capture reagent is provided, theanti-polypeptide capture reagent itself can be labelled with adetectable marker, e.g. a chemiluminescent, enzymatic, fluorescent, orradioactive moiety.

The SC6 polypeptides also find use in raising antibodies. In anotheraspect, the present invention provides antibodies, which bind to an SC6polypeptide. Preferred antibodies bind specifically to SC6 polypeptidesso that they can be used to purify, capture and/or inhibit the activityof such SC6 polypeptides. The antibodies may be monoclonal, polyclonal,chimeric, humanised or bispecific, or conjugated to a therapeuticmoiety, second antibody or a fragment thereof, a cytotoxic agent orcytokine. Preferably these antibodies are human or human-chimericantibodies.

As indicated above, SC6 polypeptides represent a target for therapeuticintervention in hypoxia related conditions. Furthermore, the inventionprovides assays for use in drug discovery in order to identify or verifythe efficacy of agents for treatment or prevention of hypoxia relatedconditions. Candidate agents can be assayed for their ability tomodulate levels of an SC6 polypeptide in a subject suffering from ahypoxia related condition, e.g. cancer. Preferably, the candidate agentinhibits expression or activity levels of an SC6 polypeptide. Agentsable to modulate levels of an SC6 polypeptide in a subject sufferingfrom a hypoxia related condition, e.g. cancer, towards levels found insubjects free from any hypoxia related conditions or to produce similarchanges in an experimental model of hypoxia related conditions, can beused as lead agents for drug discovery, or used therapeutically.Expression of an SC6 polypeptide can be assayed by, for exampleimmunoassays, gel electrophoresis followed by visualization, detectionof mRNA or polypeptide activity or any other method taught herein orknown to those skilled in the art. Such assays can be used to screencandidate agents, in clinical monitoring or in drug development, whereabundance of an SC6 polypeptide can serve as a surrogate marker forclinical disease.

Hence, another aspect of the invention provides methods of screening foragents that modulate (e.g. up-regulate, down-regulate, stimulate orinhibit) the expression (e.g. protein expression) or activity (e.g.binding/transporter activity) of an SC6 polypeptide. Accordingly, theinvention provides a method of screening for agents that modulate

-   -   (i) the expression or activity of an SC6 polypeptide, or    -   (ii) the expression of an SC6 nucleic acid molecule, comprising    -   comparing the expression or activity of said polypeptide, or the        expression of said nucleic acid molecule, in the presence of a        candidate agent with the expression or activity of said        polypeptide, or the expression of said nucleic acid molecule, in        the absence of the candidate agent or in the presence of a        control agent; and determining whether the candidate agent        causes the expression or activity of said polypeptide, or the        expression of said nucleic acid molecule, to change.

In one embodiment, the expression or activity level of said polypeptide,or the expression level of said nucleic acid molecule is compared with apredetermined reference range. Preferably, the agents down-regulate orinhibit, the expression, e.g. protein expression, or activity, e.g.binding/transporter activity, of an SC6 polypeptide, or the expressionof an SC6 nucleic acid molecule.

In yet another aspect, the invention provides a method of screening foragents that interact with (e.g. bind to) an SC6 polypeptide, comprisingcontacting said polypeptide with a candidate agent and determiningwhether or not the candidate agent interacts with said polypeptide.Agents that interact with an SC6 polypeptide may be used for thepreparation of therapeutic drug conjugates.

The screening assays may comprise, a growth assay (under hypoxicconditions), a binding assay or a translation inhibition assay. Bindingassays include competitive binding assays, wherein the binding affinityof the candidate compound is compared with that of a known enzymesubstrate for SC6, a preferred enzyme substrate is taurine.

Examples of candidate agents which may be tested for activity in thedescribed assays, include, but are not limited to, nucleic acids (e.g.DNA and RNA), carbohydrates, lipids, proteins, peptides,peptidomimetics, agonists, antagonists, small molecules and other drugs.Agents can be obtained using any of the numerous suitable approaches incombinatorial library methods known in the art, including: biologicallibraries; spatially addressable parallel solid phase or solution phaselibraries; synthetic library methods requiring deconvolution; the“one-bead one-compound” library method; and synthetic library methodsusing affinity chromatography selection. The biological library approachis limited to peptide libraries, while the other four approaches areapplicable to peptide, non-peptide oligomer or small molecule librariesof compounds (Lam, 1997, Anticancer Drug Des. 12:145; U.S. Pat. No.5,738,996 and U.S. Pat. No. 5,807,683.

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al., 1993, Proc. Natl. Acad.Sci. USA 90:6909; Erb et al., 1994, Proc. Natl. Acad. Sci. USA 91:11422;Zuckermann et al., 1994, J. Med. Chem. 37:2678; Cho et al., 1993,Science 261:1303; Carrell et al., 1994, Angew. Chem. Int. Ed. Engl.33:2059; Carell et al., 1994, Angew. Chem. Int. Ed. Engl. 33:2061; andGallop et al., 1994, J. Med. Chem. 37:1233.

Libraries of compounds may be presented, e.g. presented in solution(e.g. Houghten, 1992, Bio/Techniques 13:412-421), or on beads (Lam,1991, Nature 354:82-84), chips (Fodor, 1993, Nature 364:555-556),bacteria (U.S. Pat. No. 5,223,409), spores (U.S. Pat. No. 5,571,698;U.S. Pat. No. 5,403,484; and U.S. Pat. No. 5,223,409), plasmids (Cull etal., 1992, Proc. Natl. Acad. Sci. USA 89:1865-1869) or phage (Scott andSmith, 1990, Science 249:386-390; Devlin, 1990, Science 249:404-406;Cwirla et al., 1990, Proc. Natl. Acad. Sci. USA 87:6378-6382; andFelici, 1991, J. Mol. Biol. 222:301-310).

In one embodiment, agents that interact with (i.e. bind to) an SC6polypeptide (or nucleic acid) are identified in a cell-based assaysystem. In accordance with this embodiment, cells expressing an SC6polypeptide (or nucleic acid) are contacted with a candidate agent or acontrol agent and the ability of the candidate agent to interact withthe SC6 polypeptide (or nucleic acid) is determined. If desired, thisassay may be used to screen a plurality (e.g. a library) of candidateagents. The cell, for example, can be of prokaryotic origin (e.g. E.coli) or eukaryotic origin (e.g. yeast or mammalian). Further, the cellscan express the SC6 polypeptide endogenously or be geneticallyengineered to express said polypeptide. In some embodiments, the SC6polypeptide (or nucleic acid) or the candidate agent is labelled, forexample with a radioactive label (such as ³H, ³²P, ³⁵S or ¹²⁵I) or afluorescent label (such as fluorescein isothiocyanate, rhodamine,phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde orfluorescamine) to enable detection of an interaction between saidpolypeptide (or nucleic acid) and a candidate agent. The ability of thecandidate agent to interact directly or indirectly with the SC6polypeptide (or nucleic acid) can be determined by methods known tothose of skill in the art. For example, the interaction between acandidate agent and an SC6 polypeptide can be determined by flowcytometry, a scintillation assay, immunoprecipitation or western blotanalysis.

In another embodiment, agents that interact with (i.e. bind to) an SC6polypeptide (or nucleic acid) are identified in a cell-free assaysystem. In accordance with this embodiment, a native or recombinant SC6polypeptide (or nucleic acid) or fragment thereof, is contacted with acandidate agent or a control agent and the ability of the candidateagent to interact with the polypeptide is determined. If desired, thisassay may be used to screen a plurality (e.g. a library) of candidateagents. Preferably, the SC6 polypeptide (or nucleic acid) is firstimmobilized, by, for example, contacting the SC6 polypeptide with animmobilized antibody which specifically recognizes and binds it, or bycontacting a purified preparation of polypeptide with a surface designedto bind proteins, or by contacting a purified preparation of nucleicacid molecules with a surface designed to bind nucleic acids. The SC6polypeptide (or nucleic acid) may be partially or completely purified(e.g. partially or completely free of other polypeptides or nucleicacids) or part of a cell lysate. Further, the SC6 polypeptide may be afusion protein comprising the SC6 polypeptide for use in the inventionor a biologically active portion thereof and a domain such asglutathionine-S-transferase. Alternatively, the SC6 polypeptide can bebiotinylated using techniques well known to those of skill in the art(e.g. biotinylation kit, Pierce Chemicals; Rockford, Ill., USA). Theability of the candidate agent to interact with the SC6 polypeptide canbe can be determined by methods known to those of skill in the art.

In another embodiment, a cell-based assay system is used to identifyagents that bind to or modulate the activity of a protein, such as anenzyme, or a biologically active portion thereof, which is responsiblefor the production or degradation of the SC6 polypeptide or isresponsible for the post-translational modification of the polypeptide.In a primary screen, a plurality (e.g. a library) of agents arecontacted with cells that naturally or recombinantly express: (i) an SC6polypeptide and (ii) a protein that is responsible for processing of theSC6 polypeptide, in order to identify compounds that modulate theproduction, degradation, or post-translational modification of the SC6polypeptide. If desired, agents identified in the primary screen canthen be assayed in a secondary screen against cells naturally orrecombinantly expressing the SC6 polypeptide. The ability of thecandidate agent to modulate the production, degradation orpost-translational modification of an SC6 polypeptide can be determinedby methods known to those of skill in the art, including withoutlimitation, flow cytometry, a scintillation assay, immunoprecipitationand western blot analysis.

In another embodiment, agents that competitively interact with (e.g.bind to) an SC6 polypeptide are identified in a competitive bindingassay. In accordance with this embodiment, cells expressing thepolypeptide are contacted with a candidate agent and an agent known tointeract with the polypeptide, e.g. taurine; the ability of thecandidate agent to competitively interact with the polypeptide is thendetermined. Alternatively, agents that competitively interact with (i.e.bind to) an SC6 polypeptide are identified in a cell-free assay systemby contacting the polypeptide with a candidate agent and an agent knownto interact with the polypeptide, e.g. taurine. As stated above, theability of the candidate agent to interact with an SC6 polypeptide canbe determined by methods known to those of skill in the art. Theseassays, whether cell-based or cell-free, can be used to screen aplurality (e.g. a library) of candidate agents.

In another embodiment, agents that modulate (i.e. upregulate ordownregulate) the expression of an SC6 polypeptide (or nucleic acid) areidentified by contacting cells (e.g. cells of prokaryotic or eukaryoticorigin) expressing the SC6 polypeptide (or nucleic acid) with acandidate agent or a control agent (e.g. phosphate buffered saline(PBS)) and determining the expression of the SC6 polypeptide (or nucleicacid e.g. mRNA) encoding the polypeptide. The level of expression of aselected SC6 polypeptide or mRNA encoding an SC6 polypeptide in thepresence of the candidate agent is compared to the level of expressionof the SC6 polypeptide or mRNA encoding the SC6 polypeptide in theabsence of the candidate agent (e.g. in the presence of a controlagent). The candidate agent can then be identified as a modulator of theexpression of the SC6 polypeptide based on this comparison. For example,when expression of the SC6 polypeptide or mRNA encoding the SC6polypeptide is significantly greater in the presence of the candidateagent than in its absence, the candidate agent is identified as astimulator of expression of the SC6 polypeptide or mRNA encoding the SC6polypeptide. Alternatively, when expression of the SC6 polypeptide ormRNA encoding the SC6 polypeptide is significantly less in the presenceof the candidate agent than in its absence, the candidate agent isidentified as an inhibitor of the expression of the polypeptide or mRNAencoding the polypeptide. The level of expression, of an SC6 polypeptide(or nucleic acid) can be determined by methods known to those of skillin the art based on the present description. For example, mRNAexpression can be assessed by Northern blot analysis or RT-PCR, andprotein levels can be assessed by western blot analysis.

In another embodiment, agents that modulate the activity of an SC6polypeptide for use in the invention are identified by contacting apreparation containing the SC6 polypeptide, or cells (e.g. prokaryoticor eukaryotic cells) expressing the SC6 polypeptide with a candidateagent or a control agent and determining the ability of the candidateagent to modulate (e.g. stimulate or inhibit) the activity of the SC6polypeptide. Preferably the candidate agent inhibits the activity of theSC6 polypeptide. The activity of an SC6 polypeptide can be assessed bydetecting its effect on a “downstream effector” or substrate, forexample, but without limitation, induction of a cellular signaltransduction pathway of the polypeptide (e.g. intracellular Ca²⁺,diacylglycerol, IP3, etc.), detecting catalytic or enzymatic activity ofthe target on a suitable substrate (e.g. taurine concentrations, Shi Y Ret al., (2000) Acta Pharmacol Sin, October 23, 10, 910-918 & Warskulat Uet al., (1997) 321, 683-690), detecting the induction of a reporter gene(e.g. a regulatory element that is responsive to a polypeptide of theinvention and is operably linked to a nucleic acid encoding a detectablemarker, e.g. luciferase), or detecting a cellular response, for example,cellular differentiation, or cell proliferation as the case may be,based on the present description, techniques known to those of skill inthe art can be used for measuring these activities (see, e.g. U.S. Pat.No. 5,401,639). The candidate agent can then be identified as amodulator of the activity of an SC6 polypeptide by comparing the effectsof the candidate agent to the control agent. Suitable control agentsinclude phosphate buffered saline (PBS) and normal saline (NS).

In another embodiment, agents that modulate (e.g. upregulate ordownregulate) the expression, activity or both the expression andactivity of an SC6 polypeptide for use in the invention are identifiedin an animal model. Examples of suitable animals include, but are notlimited to, mice, rats, rabbits, monkeys, guinea pigs, dogs and cats. Inaccordance with this embodiment, the candidate agent or a control agentis administered (e.g. orally, rectally or parenterally such asintraperitoneally or intravenously) to a suitable animal and the effecton the expression, activity or both expression and activity of thepolypeptide is determined. Changes in the expression of an SC6polypeptide for use in the invention can be assessed by any suitablemethod described above, based on the present description.

In yet another embodiment, an SC6 polypeptide for use in the inventionis used as a “bait protein” in a two-hybrid assay or three hybrid assayto identify other proteins that bind to or interact with the SC6polypeptide (see, e.g. U.S. Pat. No. 5,283,317; Zervos et al. (1993)Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054;Bartel et al. (1993) Bio/Techniques 14:920-924; Iwabuchi et al. (1993)Oncogene 8:1693-1696; and WO 94/10300). As those skilled in the art willappreciate, such binding proteins are also likely to be involved in thepropagation of signals by the SC6 polypeptides for use in the inventionas, for example, upstream or downstream elements of a signalling pathwayinvolving the SC6 polypeptides for use in the invention.

One skilled in the art will appreciate that an SC6 polypeptide may beused in a method for the structure-based design of an agent, inparticular a small molecule which acts to modulate (e.g. stimulate orinhibit) the activity of said polypeptide, said method comprising:

-   -   1) determining the three-dimensional structure of said        polypeptide,    -   2) deducing the three-dimensional structure of the likely        reactive or binding site(s) of said polypeptide,    -   3) synthesising candidate agents that are predicted to react or        bind to the deduced reactive or binding site, and    -   4) testing whether the candidate agent is able to modulate the        activity of said polypeptide.

It will be appreciated that the above process is likely to be aniterative process. In a preferred embodiment the agent inhibits an SC6polypeptide. In another preferred embodiment the agent binds to anallosteric binding site on the SC6 polypeptide and inhibits SC6polypeptide activity.

Thus, the invention further provides agents identified by theabove-described screening assays and uses thereof for treatments asdescribed herein. In addition, the invention also provides the use of anagent which interacts with, or modulates the expression and/or activityof an SC6 polypeptide, in the manufacture of a medicament for thetreatment of hypoxia related conditions, e.g. cancer. Preferably, theagent interacts with or inhibits the expression and/or activity of anSC6 polypeptide. Such agents may be used in the manufacture of amedicament for the treatment of hypoxia related conditions, e.g. cancer.When a reference is made herein to a method of treating or preventing adisease or condition using a particular agent or combination of agents,it is to be understood that such a reference is intended to include theuse of that agent or combination of agents in the preparation of amedicament (pharmaceutical composition) for the treatment or preventionof the disease or condition.

As discussed herein agents of the invention find use in the treatment orprophylaxis of hypoxia related conditions e.g. cancer. Thus, in anotheraspect, the present invention provides a pharmaceutical compositioncomprising at least one agent, optionally together with one or morepharmaceutically acceptable excipients, carriers or diluents. In anotheraspect of the invention the pharmaceutical composition is for use as avaccine and so any additional components will be acceptable for vaccineuse. In addition, the skilled person will appreciate that one or moresuitable adjuvants may be added to such vaccine preparations.

The pharmaceutical composition can be administered simultaneously,separately or sequentially with another appropriate treatment forhypoxia related conditions, such as but not limited to, cancer,angiogenesis, and other angiogenesis related disorders, such as but notlimited to, diabetic retinopathy, asthma, macular degeneration,psoriasis and rheumatoid arthritis.

The pharmaceutical composition may be administered to a subject by anyof the routes conventionally used for drug administration, for examplethey may be adapted for oral (including buccal, sublingual), topical(including transdermal), nasal (including inhalation), rectal, vaginalor parenteral (including subcutaneous, intramuscular, intravenous orintradermal) administration to mammals including humans. The mostsuitable route for administration in any given case will depend on theparticular compound or pharmaceutical composition, the subject, and thenature and severity of the disease and the physical condition of thesubject. Such compositions may be prepared by any method known in theart of pharmacy, for example by bringing into association the activeagent with the carrier(s), excipient(s) or diluents.

The composition will usually be supplied as part of a sterile,pharmaceutical composition that will normally include a pharmaceuticallyacceptable carrier. This pharmaceutical composition may be in anysuitable form, (depending upon the desired method of administering it toa patient).

Pharmaceutical compositions adapted for oral administration may bepresented as discrete units such as capsules or tablets, powders orgranules, solutions or suspensions in aqueous or non-aqueous liquids,edible foams or whips, or oil-in-water liquid emulsions or water-in-oilliquid emulsions.

Tablets and capsules for oral administration may be in unit dosepresentation form, and may contain conventional excipients such asbinding agents, for example syrup, acacia, gelatin, sorbitol,tragacanth, or polyvinylpyrrolidone; fillers, for example lactose,sugar, maize-starch, calcium phosphate, sorbitol or glycine; tablettinglubricants, for example magnesium stearate, talc, polyethylene glycol orsilica; disintegrants, for example potato starch; or acceptable wettingagents such as sodium lauryl sulfate. The tablets may be coatedaccording to methods well known in normal pharmaceutical practice. Oralliquid preparations may be in the form of, for example, aqueous or oilysuspensions, solutions, emulsions, syrups or elixirs, or may bepresented as a dry product for reconstitution with water or othersuitable vehicle before use. Such liquid preparations may containconventional additives, such as suspending agents, for example sorbitol,methyl cellulose, glucose syrup, gelatin, hydroxyethyl cellulose,carboxymethyl cellulose, aluminium stearate gel or hydrogenated ediblefats, emulsifying agents, for example lecithin, sorbitan monooleate, oracacia; non-aqueous vehicles (which may include edible oils), forexample almond oil, oily esters such as glycerine, propylene glycol, orethyl alcohol; preservatives, for example methyl or propylp-hydroxybenzoate or sorbic acid, and, if desired, conventionalflavouring or colouring agents.

Pharmaceutical compositions adapted for topical administration may beformulated as ointments, creams, suspensions, lotions, powders,solutions, pastes, gels, impregnated dressings, sprays, aerosols or oilsand may contain appropriate conventional additives such aspreservatives, solvents to assist drug penetration and emollients inointments and creams. Such applications include those to the eye orother external tissues, for example the mouth and skin and thecompositions are preferably applied as a topical ointment or cream. Whenformulated in an ointment, the active agent may be employed with eithera paraffinic or a water-miscible ointment base. Alternatively, theactive agent may be formulated in a cream with an oil-in-water creambase or a water-in-oil base. The composition may also contain compatibleconventional carriers, such as cream or ointment bases and ethanol oroleyl alcohol for lotions.

Pharmaceutical compositions adapted for topical administration to theeye include eye drops wherein the active agent is dissolved or suspendedin a suitable carrier, especially an aqueous solvent.

Pharmaceutical compositions adapted for topical administration in themouth include lozenges, pastilles and mouth washes.

Pharmaceutical compositions adapted for transdermal administration maybe presented as discrete patches intended to remain in intimate contactwith the epidermis of the recipient for a prolonged period of time. Forexample, the active agent may be delivered from the patch byiontophoresis as generally described in Pharmaceutical Research, 3(6),318, (1986).

Pharmaceutical compositions adapted for nasal administration wherein thecarrier is a solid include a coarse powder having a particle size forexample in the range 20 to 500 microns which is administered by rapidinhalation through the nasal passage from a container of the powder heldclose up to the nose. Suitable compositions wherein the carrier is aliquid, for administration as a nasal spray or as nasal drops, includeaqueous or oil solutions of the active ingredient.

Pharmaceutical compositions adapted for administration by inhalationinclude fine particle dusts or mists which may be generated by means ofvarious types of metered dose pressurised aerosols, nebulizers orinsufflators.

Pharmaceutical compositions adapted for rectal administration may bepresented as suppositories or enemas. Suppositories will containconventional suppository bases, e.g. cocoa-butter or other glyceride.

Pharmaceutical compositions adapted for vaginal administration may bepresented as pessaries, tampons, creams, gels, pastes, foams or spraycompositions.

Pharmaceutical compositions adapted for parenteral administrationinclude aqueous and non-aqueous sterile injection solutions which maycontain anti-oxidants, buffers, bacteriostats and solutes which renderthe formulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. The compositions may be presented inunit-dose or multi-dose containers, for example sealed ampoules andvials, and may be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of the sterile liquid carrier, for examplewater for injections, immediately prior to use. Extemporaneous injectionsolutions and suspensions may be prepared from sterile powders, granulesand tablets.

For parenteral administration, fluid unit dosage forms are preparedutilizing the active agent and a sterile vehicle, water being preferred.The active ingredient, depending on the vehicle and concentration used,can be either suspended or dissolved in the vehicle. In preparingsolutions the active agent can be dissolved in water for injection andfilter sterilised before filling into a suitable vial or ampoule andsealing.

Advantageously, agents such as a local anaesthetic, preservative andbuffering agents can be dissolved in the vehicle. To enhance thestability, the composition can be frozen after filling into the vial andthe water removed under vacuum. The dry lyophilized powder is thensealed in the vial and an accompanying vial of water for injection maybe supplied to reconstitute the liquid prior to use. Parenteralsuspensions are prepared in substantially the same manner except thatthe active agent is suspended in the vehicle instead of being dissolvedand sterilization cannot be accomplished by filtration. The active agentcan be sterilised by exposure to ethylene oxide before suspending in thesterile vehicle. Advantageously, a surfactant or wetting agent isincluded in the composition to facilitate uniform distribution of theactive ingredient.

It should be understood that in addition to the ingredients particularlymentioned above, the compositions may also include other agentsconventional in the art having regard to the type of formulation inquestion, for example those suitable for oral administration may includeflavouring agents. They may also contain therapeutically active agentsin addition to the agents of the present invention. Such carriers may bepresent as from about 1% w/w up to about 98% w/w of the formulation.More usually they will form up to about 80% of the formulation.

The compositions may contain from 0.1% by weight, preferably from 10-60%by weight, of the active material, depending on the method ofadministration.

The pharmaceutical compositions may be provided in unit dosage form,will generally be provided in a sealed container and may be provided aspart of a kit. Such a kit would normally (although not necessarily)include instructions for use. It may include a plurality of said unitdosage forms. Preferred unit dosage compositions are those containing adaily dose or sub-dose, or an appropriate fraction thereof, of an activeingredient.

Optimal dosages of the pharmaceutical compositions can vary between widelimits, depending upon the nature and extent of the condition beingtreated, the form, route and site of administration, and the particularsubject being treated, and such optimums can be determined byconventional techniques. It will also be appreciated by one of skill inthe art that the optimal course of treatment, i.e. the number of dosesof the aforementioned compositions given per day for a defined number ofdays, can be ascertained by those skilled in the art using conventionalcourse of treatment determination tests. If side effects develop theamount and/or frequency of the dosage can be reduced, in accordance withnormal clinical practice.

In additional aspects, the present invention provides,

-   i) a method for the prophylaxis and/or treatment of a subject    suffering from a hypoxia related condition, which comprises    administering to said subject a therapeutically effective amount of    at least one SC6 polypeptide or an SC6 nucleic acid as described    herein.-   ii) a method for the prophylaxis and/or treatment of a subject    suffering from a hypoxia related condition, which comprises    administering to said subject a therapeutically effective amount of    an agent that inhibits the expression or activity of an SC6    polypeptide as described herein.-   iii) the use of at least one SC6 polypeptide or an SC6 nucleic acid,    as described herein, for the prophylaxis and/or treatment of hypoxia    related conditions.-   iv) the use of an agent that inhibits the expression or activity of    an SC6 polypeptide, as described herein, for the prophylaxis and/or    treatment of hypoxia related conditions.-   v) the use of at least one SC6 polypeptide or SC6 nucleic acid, as    described herein, in the preparation of a medicament for use in the    prophylaxis and/or treatment of hypoxia related conditions.-   vi) the use of an agent that inhibits the expression or activity of    an SC6 polypeptide, as described herein, in the preparation of a    medicament for use in the prophylaxis and/or treatment of hypoxia    related conditions.

Preferably, the agent for use, in the method or use of the additionalaspects described above, is an antibody that binds to an SC6polypeptide.

In view of the importance of SC6 in cancer the following form furtheradditional aspects of the present invention:

-   vii) a method for monitoring/assessing cancer treatment, such as    cervical, colon, renal, lung, uterine, breast or pancreatic cell    carcinoma, lymphoma, e.g. Burkitt's lymphoma, or leukaemia, e.g.    myeloid or T-cell leukaemia, treatment in a patient, which comprises    the step of determining the presence or absence and/or quantifying    at least one SC6 polypeptide in a biological sample obtained from    said patient.-   viii) a method for the identification of hypoxic cancer cells, such    as hypoxic cervical, colon, renal, lung, uterine, breast or    pancreatic cell carcinoma, lymphoma, e.g. Burkitt's lymphoma; or    leukaemia, e.g. myeloid or T-cell leukaemia cells, in a biological    sample obtained from a subject, which comprises the step of    determining the presence or absence and/or quantifying at least one    SC6 polypeptide.

In order to more fully appreciate the present invention, SC6polypeptides will now be discussed in greater detail.

The term “polypeptides” includes peptides, polypeptides and proteins.These are used interchangeably unless otherwise specified.

The polypeptides are preferably provided in substantially pure form,that is to say, they are free, to a substantial extent, from otherproteins. Thus, an SC6 polypeptide for use in the present invention maybe provided in a composition in which it is the predominant componentpresent (i.e. it is present at a level of at least 50%; preferably atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95%;when determined on a weight/weight basis excluding solvents orcarriers).

SC6 polypeptides for use in the present invention may be in isolated orrecombinant form, and may be fused to other moieties. SC6 polypeptidesmay be in the form of a “mature” protein or may be part of a largerprotein such as a fusion protein. It is often advantageous to include anadditional amino acid sequence which contains secretory or leadersequences, a pre-, pro- or prepro-protein sequence, or a sequence whichaids in purification such as an affinity tag, for example, but withoutlimitation, multiple histidine residues, a FLAG tag, HA tag or myc tag.An additional sequence which may provide stability during recombinantproduction may also be used. Such sequences may be optionally removed asrequired by incorporating a cleavable sequence as an additional sequenceor part thereof. Thus, an SC6 polypeptide may be fused to other moietiesincluding other polypeptides. Such additional sequences and affinitytags are well known in the art.

In particular, fusions of the SC6 polypeptides withlocalisation-reporter proteins, such as, the Green Fluorescent Protein(U.S. Pat. No. 5,625,048, U.S. Pat. No. 5,777,079, U.S. Pat. No.6,054,321 and U.S. Pat. No. 5,804,387), the DsRed fluorescent protein(Matz, M. V. et al., (1999) Nature Biotech. 17:969-973) or fluorescentproteins from nonbioluminescent Anthozoa species, are specificallycontemplated by the present invention.

Polypeptides within the Scope of a)

A polypeptide within the scope of a), may consist of the particularamino acid sequence of SEQ ID No. 1 or may have an additional N-terminaland/or an additional C-terminal amino acid sequence relative to thesequence of SEQ ID No. 1.

Additional N-terminal or C-terminal sequences may be provided forvarious reasons. Techniques for providing such additional sequences arewell known in the art. Additional sequences may be provided in order toalter the characteristics of a particular polypeptide. This can beuseful in improving expression or regulation of expression in particularexpression systems. For example, an additional sequence may provide someprotection against proteolytic cleavage. This has been done for thehormone Somatostatin by fusing it at its N-terminus to part of the βgalactosidase enzyme (Itakwa et al., Science 198: 105-63 (1977)).

Additional sequences can also be useful in altering the properties of apolypeptide to aid in identification or purification. For example, afusion protein may be provided in which a polypeptide is linked to amoiety capable of being isolated by affinity chromatography. The moietymay be an antigen or an epitope and the affinity column may compriseimmobilised antibodies or immobilised antibody fragments which bind tosaid antigen or epitope (desirably with a high degree of specificity).The fusion protein can usually be eluted from the column by addition ofan appropriate buffer.

Additional N-terminal or C-terminal sequences may, however, be presentsimply as a result of a particular technique used to obtain an SC6polypeptide and need not provide any particular advantageouscharacteristic to the polypeptide. Such polypeptide are within the scopeof the present invention.

Whatever additional N-terminal or C-terminal sequence is present, it ispreferred that the resultant polypeptide should exhibit theimmunological and/or transporter activity of the polypeptide having theamino acid sequence of SEQ ID No. 1.

Polypeptides within the Scope of b)

Turning now to the polypeptides defined in b) above, it will beappreciated by the person skilled in the art that these polypeptides arevariants of the polypeptide given in a) above, provided that suchvariants exhibit the immunological and/or transporter activity of thepolypeptide having the amino acid sequence of SEQ ID No. 1.

Alterations in the amino acid sequence of a protein can occur which donot affect the function of a protein. These include amino acidsubstitutions, deletions, and insertions and can result from alternativesplicing and/or the presence of multiple translation start sites andstop sites. Polymorphisms may arise as a result of the infidelity of thetranslation process. Thus changes in amino acid sequence may betolerated which do not affect the protein's function.

The skilled person will appreciate that various changes can often bemade to the amino acid sequence of a polypeptide which has a particularactivity to produce variants (sometimes known as “muteins”) having atleast a proportion of said activity, and preferably having a substantialproportion of said activity. Such variants of the polypeptides describedin a) above are within the scope of the present invention and arediscussed in greater detail below. They include allelic and non-allelicvariants.

An example of a variant for use in the present invention is apolypeptide as defined in a) above, apart from the substitution of oneor more amino acids with one or more other amino acids. The skilledperson is aware that various amino acids have similar properties. One ormore such amino acids of a substance can often be substituted by one ormore other such amino acids without eliminating a desired activity ofthat substance. Thus, the amino acids glycine, alanine, valine, leucineand isoleucine can often be substituted for one another (amino acidshaving aliphatic side chains). Of these possible substitutions, it ispreferred that glycine and alanine are used to substitute for oneanother (since they have relatively short side chains) and that valine,leucine and isoleucine are used to substitute for one another (sincethey have larger aliphatic side chains which are hydrophobic).

Other amino acids which can often be substituted for one anotherinclude:

-   -   phenylalanine, tyrosine and tryptophan (amino acids having        aromatic side chains);    -   lysine, arginine and histidine (amino acids having basic side        chains);    -   aspartate and glutamate (amino acids having acidic side chains);    -   asparagine and glutamine (amino acids having amide side chains);        and    -   cysteine and methionine (amino acids having sulphur-containing        side chains).

Substitutions of this nature are often referred to as “conservative” or“semi-conservative” amino acid substitutions.

Amino acid deletions or insertions may also be made relative to theamino acid sequence given in a) above. Thus, for example, amino acidswhich do not have a substantial effect on the activity of thepolypeptide, or at least which do not eliminate such activity, may bedeleted. Such deletions can be advantageous since the overall length andthe molecular weight of a polypeptide can be reduced whilst stillretaining activity. This can enable the amount of polypeptide requiredfor a particular purpose to be reduced for example, dosage levels can bereduced. Amino acid insertions relative to the sequence given in a)above can also be made. This may be done to alter the properties of apolypeptide (e.g. to assist in identification, purification orexpression, as explained above in relation to fusion proteins).

Amino acid changes relative to the sequence given in a) above can bemade using any suitable technique e.g. by using site-directedmutagenesis (Hutchinson et al., (1978) J. Biol. Chem. 253:6551).

It should be appreciated that amino acid substitutions or insertionswithin the scope of the present invention can be made using naturallyoccurring or non-naturally occurring amino acids. Whether or not naturalor synthetic amino acids are used, it is preferred that only L-aminoacids are present.

In one particular embodiment, the substituted amino acid(s) dosignificantly affect the activity of the SC6 polypeptide and may beselected specifically to render dominant negative activity upon thepeptide. In another embodiment, the substituted amino acid(s) may beselected specifically to render the polypeptide constitutively active.

Modifications include naturally occurring modifications such as andwithout limitation, post-translational modifications and alsonon-naturally occurring modifications such as may be introduced bymutagenesis.

Whatever amino acid changes are made (whether by means of substitution,insertion, deletion or modification), preferred SC6 polypeptides have atleast 50% sequence identity with the polypeptide of SEQ ID No.1, morepreferably the degree of sequence identity is at least 75%. Yet morepreferably the degree of sequence identity is at least 80% or at least85%. Sequence identities of at least 90%, or at least 95%, or at least98% are most preferred.

Percentage identity is a well known concept in the art and is determinedfor two amino acid or nucleic acid sequences by aligning the sequencesfor optimal comparison purposes (e.g. gaps can be introduced in thefirst sequence for best alignment with the sequence) and comparing theamino acid residues or nucleotides at corresponding positions. The “bestalignment” is an alignment of two sequences which results in the highestpercent identity. The percent identity is determined by the number ofidentical amino acid residues or nucleotides in the sequences beingcompared, i.e. Percentage identity=no. of identical positions/total no.of positions×100.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm known to those of skill inthe art. An example of a mathematical algorithm for comparing twosequences is the algorithm of Karlin and Altschul (1990) Proc. Natl.Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5877. The NBLAST and XBLAST programsof Altschul, et al. (1990) J. Mol. Biol. 215:403-410 have incorporatedsuch an algorithm. BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to proteinmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilised as described in Altschul et al.(1997) Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can beused to perform an iterated search which detects distant relationshipsbetween molecules (Id.). When utilising BLAST, Gapped BLAST, andPSI-Blast programs, the default parameters of the respective programs(e.g. XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

Another example of a mathematical algorithm utilised for the comparisonof sequences is the algorithm of Myers & Miller, CABIOS (1989). TheALIGN program (version 2.0) which is part of the CGC sequence alignmentsoftware package has incorporated such an algorithm. Other algorithmsfor sequence analysis known in the art include ADVANCE and ADAM asdescribed in Torellis & Robotti (1994) Comput. Appl. Biosci., 10:3-5;and FASTA described in Pearson & Lipman (1988) Proc. Natl. Acad. Sci.85:2444-8. Within FASTA, ktup is a control option that sets thesensitivity and speed of the search. Where high degrees of sequenceidentity are present there will be relatively few differences in aminoacid sequence. Thus for example they may be less than 20, less than 10,or even less than 5 differences.

Polypeptides within the Scope of c)

As discussed supra, it is often advantageous to reduce the length of apolypeptide, provided that the resultant reduced length polypeptidestill has a desired activity or can give rise to useful antibodies or avaccine. Feature c) therefore covers fragments of polypeptides a) or b)above.

The skilled person can determine whether or not a particular fragmenthas activity using the techniques disclosed above. Preferred fragmentsare at least 10, at least 20, at least 50 or at least 100 amino acidslong.

SC6 polypeptides will find use in a therapeutic approach to hypoxiarelated conditions, particularly for cancer. The skilled person willappreciate that for the preparation of one or more polypeptides for usein the invention, the preferred approach will be based on recombinantDNA techniques.

SC6 polypeptides can be coded for by a large variety of nucleic acidmolecules, taking into account the well-known degeneracy of the geneticcode. All of these molecules are within the scope of the presentinvention. They can be inserted into vectors and cloned to provide largeamounts of DNA or RNA for further study. Suitable vectors may beintroduced into host cells to enable the expression of SC6 polypeptidesusing techniques known to the person skilled in the art.

The term “RNA equivalent” when used above indicates that a given RNAmolecule has a sequence which is complementary to that of a given DNAmolecule, allowing for the fact that in RNA “U” replaces “T” in thegenetic code. The nucleic acid molecule may be in isolated, recombinantor chemically synthetic form.

Techniques for cloning, expressing and purifying polypeptides are wellknown to the skilled person. DNA constructs can readily be generatedusing methods well known in the art. These techniques are disclosed, forexample in J. Sambrook et al, Molecular Cloning 2^(nd) Edition, ColdSpring Harbour Laboratory Press (1989); in Old & Primrose Principles ofGene Manipulation 5th Edition, Blackwell Scientific Publications (1994);and in Stryer, Biochemistry 4th Edition, W H Freeman and Company (1995).Modifications of DNA constructs and the proteins expressed such as theaddition of promoters, enhancers, signal sequences, leader sequences,translation start and stop signals and DNA stability controllingregions, or the addition of fusion partners may then be facilitated.

Normally the DNA construct will be inserted into a vector, which may beof phage or plasmid origin. Expression of the protein is achieved by thetransformation or transfection of the vector into a host cell which maybe of eukaryotic or prokaryotic origin. Such vectors and suitable hostcells form further aspects of the present invention.

Knowledge of the nucleic acid structure can be used to raise antibodiesand for gene therapy. Techniques for this are well-known by thoseskilled in the art.

By using appropriate expression systems, SC6 polypeptides may beexpressed in glycosylated or non-glycosylated form. Non-glycosylatedforms can be produced by expression in prokaryotic hosts, such as E.coli.

Polypeptides comprising N-terminal methionine may be produced usingcertain expression systems, whilst in others the mature polypeptide willlack this residue. Preferred techniques for cloning, expressing andpurifying a substance of the present invention are summarised below:

Polypeptides may be prepared natively or under denaturing conditions andthen subsequently refolded. Baculoviral expression vectors includesecretory plasmids (such as pACGP67 from Pharmingen), which may have anepitope tag sequence cloned in frame (e.g. myc, V5 or His) to aiddetection and allow for subsequent purification of the protein.Mammalian expression vectors may include pcDNA3 and pSecTag (bothInvitrogen), and pREP9 and pCEP4 (Invitrogen). E. coli systems includethe pBad series (His tagged-Invitrogen) or pgex series (Pharmacia).

In addition to nucleic acid molecules coding for SC6 polypeptides foruse in the present invention, referred to herein as “coding” nucleicacid molecules, the present invention also includes nucleic acidmolecules complementary thereto. Thus, for example, both strands of adouble stranded nucleic acid molecule are included within the scope ofthe present invention (whether or not they are associated with oneanother). Also included are mRNA molecules and complementary DNAMolecules (e.g. cDNA molecules).

Nucleic acid molecules that can hybridise to any of the nucleic acidmolecules discussed above are also covered by the present invention.Such nucleic acid molecules are referred to herein as “hybridising”nucleic acid molecules. Hybridising nucleic acid molecules can be usefulas probes or primers, for example.

Desirably such hybridising molecules are at least 10 nucleotides inlength and preferably are at least 25 or at least 50 nucleotides inlength. The hybridising nucleic acid molecules preferably hybridise tonucleic acids within the scope of a), b), c), d), or e), as specificallymentioned earlier above.

Desirably the hybridising molecules will hybridise to such moleculesunder stringent hybridisation conditions. One example of stringenthybridisation conditions is where attempted hybridisation is carried outat a temperature of from about 35° C. to about 65° C. using a saltsolution, which is about 0.9 molar. However, the skilled person will beable to vary such conditions as appropriate in order to take intoaccount variables such as probe length, base composition, type of ionspresent, etc.

Manipulation of the DNA encoding the protein is a particularly powerfultechnique for both modifying proteins and for generating largequantities of protein for purification purposes. This may involve theuse of PCR techniques to amplify a desired nucleic acid sequence. As afurther alternative to PCR techniques, chemical synthesis may be used.This may be automated. Relatively short sequences may be chemicallysynthesised and ligated together to provide a longer sequence.

Thus the sequence data provided herein can be used to design primers foruse in PCR so that a desired sequence can be targeted and then amplifiedto a high degree. Typically primers will be at least five nucleotideslong and will generally be at least ten nucleotides long (e.g. fifteento twenty-five nucleotides long). In some cases, primers of at leastthirty or at least thirty-five nucleotides in length may be used.

In addition to being used as primers and/or probes, hybridising nucleicacid molecules can be used as anti-sense molecules to alter theexpression of nucleic acids encoding SC6 polypeptides by binding tocomplementary nucleic acid molecules. This technique can be used inanti-sense therapy.

A hybridising nucleic acid molecule may have a high degree of sequenceidentity along its length with a nucleic acid molecule within thespecific scope of a)-e) above (e.g. at least 50%, at least 75%, at least80%, at least 85% or at least 90% or 95% sequence identity). As will beappreciated by the skilled person, the higher the sequence identity agiven single stranded nucleic acid molecule has with another nucleicacid molecule, the greater the likelihood that it will hybridise to anucleic acid molecule which is complementary to that other nucleic acidmolecule under appropriate conditions.

In view of the foregoing description the skilled person will appreciatethat a large number of nucleic acids are of use in the presentinvention. Unless the context indicates otherwise, nucleic acidmolecules of the present invention may have one or more of the followingcharacteristics:

-   1) they may be DNA or RNA;-   2) they may be single or double stranded;-   3) they may be provided in recombinant form i.e. covalently linked    to a 5′ and/or a 3′ flanking sequence to provide a molecule which    does not occur in nature;-   4) they may be provided without 5′ and/or 3′ flanking sequences    which normally occur in nature;-   5) they may be provided in substantially pure form. Thus they may be    provided in a form which is substantially free from contaminating    proteins and/or from other nucleic acids; and-   6) they may be provided with introns or without introns (e.g. as    cDNA).

The antibodies that specifically bind to one or more SC6 polypeptidesfor use in the invention are now discussed in more detail.

The SC6 polypeptide, its fragments or other derivatives, or analoguesthereof, may be used as an immunogen to generate antibodies whichimmunospecifically bind such an immunogen. Antibodies of the inventioninclude, but are not limited to monoclonal, polyclonal, chimeric,humanised or bispecific, single chain antibodies, Fab fragments andF(ab′) fragments, fragments produced by a Fab expression library,anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments ofany of the above. The antibodies may be conjugated to a therapeuticmoiety, second antibody or a fragment thereof, a cytotoxic agent orcytokine.

The term “antibody” as used herein refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin molecules, i.e.molecules that contain an antigen binding site that specifically bindsan antigen. The immunoglobulin molecules of the invention can be of anyclass (e.g. IgG, IgE, IgM, IgD and IgA) or subclass of immunoglobulinmolecule.

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art, e.g. ELISA(enzyme-linked immunosorbent assay). For example, to select antibodies,which recognize a specific domain of an SC6 polypeptide, one may assaygenerated hybridomas for a product which binds to a polypeptide fragmentcontaining such domain. For selection of an antibody that specificallybinds a first polypeptide homologue but which does not specifically bindto (or binds less avidly to) a second polypeptide homologue, one canselect on the basis of positive binding to the first polypeptidehomologue and a lack of binding to (or reduced binding to) the secondpolypeptide homologue.

For preparation of monoclonal antibodies (mAbs) directed toward an SC6polypeptide or fragment or analogue thereof, any technique whichprovides for the production of antibody molecules by continuous celllines in culture may be used. For example, the hybridoma techniqueoriginally developed by Kohler and Milstein (1975, Nature 256:495-497),as well as the trioma technique, the human B-cell hybridoma technique(Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridomatechnique to produce human monoclonal antibodies (Cole et al., 1985, inMonoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Such antibodies may be of any immunoglobulin class includingIgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridomasproducing the mAbs of the invention may be cultivated in vitro or invivo. In an additional embodiment of the invention, monoclonalantibodies can be produced in germ-free animals utilizing technologyknown in the art.

The monoclonal antibodies include but are not limited to humanmonoclonal antibodies and chimeric monoclonal antibodies (e.g.human-mouse chimeras). A chimeric antibody is a molecule in whichdifferent portions are derived from different animal species, such asthose having a human immunoglobulin constant region and a variableregion derived from a murine mAb (see, e.g. Cabilly et al., U.S. Pat.No. 4,816,567; and Boss et al., U.S. Pat. No. 4,816,397). Humanizedantibodies are antibody molecules from non-human species having one ormore complementarity determining regions (CDRs) from the non-humanspecies and a framework region from a human immunoglobulin molecule(see, e.g. Queen, U.S. Pat. No. 5,585,089).

Chimeric and humanized monoclonal antibodies can be produced byrecombinant DNA techniques known in the art, for example using methodsdescribed in WO 87/02671; EP 184,187; EP 171,496; EP 173,494; WO86/01533; U.S. Pat. No. 4,816,567; EP 125,023; Better et al., 1988,Science 240:1041-1043; Liu et al., 1987, Proc. Natl. Acad. Sci. USA84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al.,1987, Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al., 1987,Canc. Res. 47:999-1005; Wood et al., 1985, Nature 314:446-449; and Shawet al., 1988, J. Nad. Cancer Inst. 80:1553-1559; Morrison, 1985, Science229:1202-1207; Oi et al., 1986, Bio/Techniques 4:214; U.S. Pat. No.5,225,539; Jones et al., 1986, Nature 321:552-525; Verhoeyan et al.(1988) Science 239:1534; and Beidler et al., 1988, J. Immunol.141:4053-4060.

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Such antibodies can be produced usingtransgenic mice which are incapable of expressing endogenousimmunoglobulin heavy and light chain genes, but which can express humanheavy and light chain genes. The transgenic mice are immunized in thenormal fashion with a selected antigen, e.g. all or a portion of an SC6polypeptide. Monoclonal antibodies directed against the antigen can beobtained using conventional hybridoma technology. The humanimmunoglobulin transgenes harboured by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g. U.S. Pat. No. 5,625,126; U.S. Pat. No. 5,633,425; U.S. Pat. No.5,569,825; U.S. Pat. No. 5,661,016; and U.S. Pat. No. 5,545,806. Inaddition, companies such as Abgenix, Inc. (Freemont, Calif., USA) andGenpharm (San Jose, Calif., USA) can be engaged to provide humanantibodies directed against a selected antigen using technology similarto that described above.

Completely human antibodies, which recognize a selected epitope, can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g. a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope (Jespers et al. (1994) Bio/technology12:899-903).

The antibodies of the present invention can also be generated usingvarious phage display methods known in the art. In phage displaymethods, functional antibody domains are displayed on the surface ofphage particles which carry the polynucleotide sequences encoding them.In a particular, such phage can be utilized to display antigen bindingdomains expressed from a repertoire or combinatorial antibody library(e.g. human or murine). Phage expressing an antigen binding domain thatbinds the antigen of interest can be selected or identified withantigen, e.g. using labelled antigen or antigen bound or captured to asolid surface or bead. Phage used in these methods are typicallyfilamentous phage including fd and M13 binding domains expressed fromphage with Fab, Fv or disulfide stabilized Fv antibody domainsrecombinantly fused to either the phage gene III or gene vm protein.Phage display methods that can be used to make the antibodies of thepresent invention include those disclosed in Brinkman et al., J.Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods184:177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24:952-958(1994); Persic et al., Gene 187 9-18 (1997); Burton et al., Advances inImmunology 57:191-280 (1994); WO 90/02809; WO 91/10737; WO 92/01047; WO92/18619; WO 93/11236; WO 95/15982; WO 95/20401; U.S. Pat. No. 698,426;U.S. Pat. No. 5,223,409; U.S. Pat. No. 5,403,484; U.S. Pat. No.5,580,717; U.S. Pat. No. 5,427,908; U.S. Pat. No. 5,750,753; U.S. Pat.No. 5,821,047; U.S. Pat. No. 5,571,698; U.S. Pat. No. 5,427,908; U.S.Pat. No. 5,516,637; U.S. Pat. No. 5,780,225; U.S. Pat. No. 5,658,727;U.S. Pat. No. 5,733,743 and U.S. Pat. No. 5,969,108.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g. as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in WO92/22324; Mullinax et al., (1992) BioTechniques 12(6):864-869; and Sawaiet al., (1995) AJRI 34:26-34; and Better et al., (1988) Science240:1041-1043.

Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. No. 4,946,778 and U.S.Pat. No. 5,258,498; Huston et al., (1991) Methods in Enzymology203:46-88; Shu et al., (1993) PNAS 90:7995-7999; and Skerra et al.,(1988) Science 240:1038-1040.

The invention further provides for the use of bispecific antibodies,which can be made by methods known in the art. Traditional production offull-length bispecific antibodies is based on the coexpression of twoimmunoglobulin heavy chain-light chain pairs, where the two chains havedifferent specificities (Milstein et al., 1983, Nature 305:537-539).Because of the random assortment of immunoglobulin heavy and lightchains, these hybridomas (quadromas) produce a potential mixture of 10different antibody molecules, of which only one has the correctbispecific structure. Purification of the correct molecule, which isusually done by affinity chromatography steps, is rather cumbersome, andthe product yields are low. Similar procedures are disclosed in WO93/08829 and in Traunecker et al., (1991) EMBO J. 10:3655-3659.

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion preferablyis with an immunoglobulin heavy chain constant domain, comprising atleast part of the hinge, CH2, and CH3 regions. It is preferred to havethe first heavy-chain constant region (CH1) containing the sitenecessary for light chain binding, present in at least one of thefusions. DNAs encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable hostorganism. This provides for great flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yields. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains in oneexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios are of noparticular significance.

In a preferred embodiment of this approach, the bispecific antibodiesare composed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails for generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology, 1986, 121:210.

The invention provides functionally active fragments, derivatives oranalogues of the anti-SC6 polypeptide immunoglobulin molecules.Functionally active means that the fragment, derivative or analogue isable to elicit anti-anti-idiotype antibodies (i.e. tertiary antibodies)that recognize the same antigen that is recognized by the antibody fromwhich the fragment, derivative or analogue is derived. Specifically, ina preferred embodiment the antigenicity of the idiotype of theimmunoglobulin molecule may be enhanced by deletion of framework and CDRsequences that are C-terminal to the CDR sequence that specificallyrecognizes the antigen. To determine which CDR sequences bind theantigen, synthetic peptides containing the CDR sequences can be used inbinding assays with the antigen by any binding assay method known in theart.

The present invention provides antibody fragments such as, but notlimited to, F(ab′)2 fragments and Fab fragments. Antibody fragmentswhich recognize specific epitopes may be generated by known techniques.F(ab′)2 fragments consist of the variable region, the light chainconstant region and the CH1 domain of the heavy chain and are generatedby pepsin digestion of the antibody molecule. Fab fragments aregenerated by reducing the disulfide bridges of the F(ab′)2 fragments.The invention also provides heavy chain and light chain dimmers of theantibodies of the invention, or any minimal fragment thereof such as Fvsor single chain antibodies (SCAs) (e.g. as described in U.S. Pat. No.4,946,778; Bird (1988) Science 242:423-42; Huston et al., (1988) Proc.Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., (1989) Nature334:544-54), or any other molecule with the same specificity as theantibody of the invention. Single chain antibodies are formed by linkingthe heavy and light chain fragments of the Fv region via an amino acidbridge, resulting in a single chain polypeptide. Techniques for theassembly of functional Fv fragments in E. coli may be used (Skerra etal., (1988) Science 242:1038-1041).

In other embodiments, the invention provides fusion proteins of theimmunoglobulins of the invention (or functionally active fragmentsthereof), for example in which the immunoglobulin is fused via acovalent bond (e.g. a peptide bond), at either the N-terminus or theC-terminus to an amino acid sequence of another protein (or portionthereof, preferably at least 10, 20 or 50 amino acid portion of theprotein) that is not the immunoglobulin. Preferably the immunoglobulin,or fragment thereof, is covalently linked to the other protein at theN-terminus of the constant domain. As stated above, such fusion proteinsmay facilitate purification, increase half-life in vivo, and enhance thedelivery of an antigen across an epithelial barrier to the immunesystem.

The immunoglobulins of the invention include analogues and derivativesthat are either modified, i.e. by the covalent attachment of any type ofmolecule as long as such covalent attachment that does not impairimmunospecific binding. For example, but not by way of limitation, thederivatives and analogues of the immunoglobulins include those that havebeen further modified, e.g. by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, etc. Additionally, the analogue orderivative may contain one or more non-classical amino acids.

The foregoing antibodies can be used in methods known in the artrelating to the localization and activity of the SC6 polypeptides, e.g.for imaging or radioimaging these polypeptides, measuring levels thereofin appropriate physiological samples, in diagnostic methods, etc. andfor radiotherapy.

The antibodies of the invention can be produced by any method known inthe art for the synthesis of antibodies, in particular, by chemicalsynthesis or by recombinant expression, and are preferably produced byrecombinant expression technique.

Recombinant expression of antibodies, or fragments, derivatives oranalogues thereof, requires construction of a nucleic acid that encodesthe antibody. If the nucleotide sequence of the antibody is known, anucleic acid encoding the antibody may be assembled from chemicallysynthesized oligonucleotides (e.g. as described in Kutmeier et al.,(1994) BioTechniques 17:242), which, briefly, involves the synthesis ofoverlapping oligonucleotides containing portions of the sequenceencoding antibody, annealing and ligation of those oligonucleotides, andthen amplification of the ligated oligonucleotides by PCR.

Alternatively, the nucleic acid encoding the antibody may be obtained bycloning the antibody. If a clone containing the nucleic acid encodingthe particular antibody is not available, but the sequence of theantibody molecule is known, a nucleic acid encoding the antibody may beobtained from a suitable source (e.g. an antibody cDNA library, or cDNAlibrary generated from any tissue or cells expressing the antibody) byPCR amplification using synthetic primers hybridisable to the 3′ and 5′ends of the sequence or by cloning using an oligonucleotide probespecific for the particular gene sequence.

If an antibody molecule that specifically recognizes a particularantigen is not available (or a source for a cDNA library for cloning anucleic acid encoding such an antibody), antibodies specific for aparticular antigen may be generated by any method known in the art, forexample, by immunizing an animal, such as a rabbit, to generatepolyclonal antibodies or, more preferably, by generating monoclonalantibodies. Alternatively, a clone encoding at least the Fab portion ofthe antibody may be obtained by screening Fab expression libraries (e.g.as described in Huse et al., (1989) Science 246:1275-1281) for clones ofFab fragments that bind the specific antigen or by screening antibodylibraries (see, e.g. Clackson et al., (1991) Nature 352:624; Hane etal., (1997) Proc. Natl. Acad. Sci. USA 94:4937).

Once a nucleic acid encoding at least the variable domain of theantibody molecule is obtained, it may be introduced into a vectorcontaining the nucleotide sequence encoding the constant region of theantibody molecule (see, e.g. WO 86/05807; WO 89/01036; and U.S. Pat. No.5,122,464). Vectors containing the complete light or heavy chain forco-expression with the nucleic acid to allow the expression of acomplete antibody molecule are also available. Then, the nucleic acidencoding the antibody can be used to introduce the nucleotidesubstitution(s) or deletion(s) necessary to substitute (or delete) theone or more variable region cysteine residues participating in anintrachain disulfide bond with an amino acid residue that does notcontain a sulfhydyl group. Such modifications can be carried out by anymethod known in the art for the introduction of specific mutations ordeletions in a nucleotide sequence, for example, but not limited to,chemical mutagenesis, in vitro site directed mutagenesis (Hutchinson etal., (1978) J. Biol. Chem. 253:6551), PCR based methods, etc.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., (1984) Proc. Natl. Acad. Sci. 81:851-855;Neuberger et al., (1984) Nature 312:604-608; Takeda et al., (1985)Nature 314:452-454) by splicing genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Asdescribed supra, a chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine mAb and a human antibodyconstant region, e.g. humanized antibodies.

Once a nucleic acid encoding an antibody molecule of the invention hasbeen obtained, the vector for the production of the antibody moleculemay be produced by recombinant DNA technology using techniques wellknown in the art. Thus, methods for preparing the protein of theinvention by expressing nucleic acid containing the antibody moleculesequences are described herein. Methods which are well known to thoseskilled in the art can be used to construct expression vectorscontaining an antibody molecule coding sequences and appropriatetranscriptional and translational control signals. These methodsinclude, for example, in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. See, for example, thetechniques described in Sambrook et al. (1990) Molecular Cloning, ALaboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. and Ausubel et al. (1998) Current Protocols in MolecularBiology, eds., John Wiley & Sons, NY.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody of the invention.

The host cells used to express a recombinant antibody of the inventionmay be either bacterial cells such as E. coli, or, preferably,eukaryotic cells, especially for the expression of whole recombinantantibody molecule. In particular, mammalian cells such as Chinesehamster ovary cells (CHO), in conjunction with a vector such as themajor intermediate early gene promoter element from humancytomegalovirus is an effective expression system for antibodies(Foecking et al., (1986) Gene 45:101; Cockett et al., (1990)Bio/Technology 8:2).

A variety of host-expression vector systems may be utilized to expressan antibody molecule of the invention. Such host-expression systemsrepresent vehicles by which the coding sequences of interest may beproduced and subsequently purified, but also represent cells which may,when transformed or transfected with the appropriate nucleotide codingsequences, express the antibody molecule of the invention in situ. Theseinclude but are not limited to microorganisms such as bacteria (e.g. E.coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing antibody codingsequences; yeast (e.g. Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g. baculovirus) containing the antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g. cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV)or transformed with recombinant plasmid expression vectors (e.g. Tiplasmid) containing antibody coding sequences; or mammalian cell systems(e.g. COS, CHO, BHK, 293, 3T3 cells) harbouring recombinant expressionconstructs containing promoters derived from the genome of mammaliancells (e.g. metallothionein promoter) or from mammalian viruses (e.g.the adenovirus late promoter; the vaccinia virus 7.5K promoter).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions comprising an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., (1983) EMBO J.2:1791), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, (1985)Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, (1989) J. Biol.Chem. 24:5503-5509); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding to amatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter). In mammalian host cells, a number ofviral-based expression systems (e.g. an adenovirus expression system)may be utilized.

As discussed above, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.glycosylation) and processing (e.g. cleavage) of protein products may beimportant for the function of the protein.

For long-term, high-yield production of recombinant antibodies, stableexpression is preferred. For example, cells lines that stably express anantibody of interest can be produced by transfecting the cells with anexpression vector comprising the nucleotide sequence of the antibody andthe nucleotide sequence of a selectable (e.g. neomycin or hygromycin),and selecting for expression of the selectable marker. Such engineeredcell lines may be particularly useful in screening and evaluation ofcompounds that interact directly or indirectly with the antibodymolecule.

The expression levels of the antibody molecule can be increased byvector amplification (for a review, see Bebbington and Hentschel, Theuse of vectors based on gene amplification for the expression of clonedgenes in mammalian cells in DNA cloning, (1987) Vol. 3. Academic Press,New York. When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., (1983) Mol. Cell. Biol.3:257).

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes both heavy and light chainpolypeptides. In such situations, the light chain should be placedbefore the heavy chain to avoid an excess of toxic free heavy chain(Proudfoot, (1986) Nature 322:52; Kohler, (1980) Proc. Natl. Acad. Sci.USA 77:2197). The coding sequences for the heavy and light chains maycomprise cDNA or genomic DNA.

Once the antibody molecule of the invention has been recombinantlyexpressed, it may be purified by any method known in the art forpurification of an antibody molecule, for example, by chromatography(e.g. ion exchange chromatography, affinity chromatography such as withprotein A or specific antigen, and sizing column chromatography),centrifugation, differential solubility, or by any other standardtechnique for the purification of proteins.

Alternatively, any fusion protein may be readily purified by utilizingan antibody specific for the fusion protein being expressed. Forexample, a system described by Janknecht et al. allows for the readypurification of non-denatured fusion proteins expressed in human celllines (Janknecht et al., (1991) Proc. Natl. Acad. Sci. USA 88:8972-897).In this system, the gene of interest is subcloned into a vacciniarecombination plasmid such that the open reading frame of the gene istranslationally fused to an amino-terminal tag consisting of sixhistidine residues. The tag serves as a matrix-binding domain for thefusion protein. Extracts from cells infected with recombinant vacciniavirus are loaded onto Ni²⁺ nitriloacetic acid-agarose columns andhistidine-tagged proteins are selectively eluted withimidazole-containing buffers.

In a preferred embodiment, antibodies of the invention or fragmentsthereof are conjugated to a diagnostic or therapeutic moiety. Theantibodies can be used for diagnosis or to determine the efficacy of agiven treatment regimen. Detection can be facilitated by coupling theantibody to a detectable substance. Examples of detectable substancesinclude various enzymes, prosthetic groups, fluorescent materials,luminescent materials, bioluminescent materials, radioactive nuclides,positron emitting metals (for use in positron emission tomography), andnonradioactive paramagnetic metal ions. See generally U.S. Pat. No.4,741,900 for metal ions which can be conjugated to antibodies for useas diagnostics according to the present invention. Suitable enzymesinclude horseradish peroxidase, alkaline phosphatase,beta-galactosidase, or acetylcholinesterase; suitable prosthetic groupsinclude streptavidin, avidin and biotin; suitable fluorescent materialsinclude umbelliferone, fluorescein, fluorescein isothiocyanate,rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride andphycoerythrin; suitable luminescent materials include luminol; suitablebioluminescent materials include luciferase, luciferin, and aequorin;and suitable radioactive nuclides include ¹²⁵I, ¹³¹I, ¹¹¹In and ⁹⁹Tc.

Antibodies of the invention or fragments thereof can be conjugated to atherapeutic agent or drug moiety to modify a given biological response.The therapeutic agent or drug moiety is not to be construed as limitedto classical chemical therapeutic agents. For example, the drug moietymay be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, a toxin such as abrin,ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such astumor necrosis factor, α-interferon, β-interferon, nerve growth factor,platelet derived growth factor, tissue plasminogen activator, athrombotic agent or an anti-angiogenic agent, e.g. angiostatin orendostatin; or, a biological response modifier such as a lymphokine,interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6),granulocyte macrophage colony stimulating factor (GM-CSF), granulocytecolony stimulating factor (G-CSF), nerve growth factor (NGF) or othergrowth factor.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g. Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (1985) eds. Alan R. Liss, Inc. pp.243-56; Hellstrom et al., “Antibodies For Drug Delivery”, in ControlledDrug Delivery (1987), eds. Robinson et al. (Marcel Dekker, Inc.) pp.623-53; Thorpe, “Antibody Carriers Of Cytotoxic Agents In CancerTherapy: A Review”, in Monoclonal Antibodies '84: Biological AndClinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985);“Analysis, Results, And Future Prospective Of The Therapeutic Use OfRadiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies ForCancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16(Academic Press 1985), Thorpe et al., “The Preparation And CytotoxicProperties Of Antibody-Toxin Conjugates”, Immunol. Rev., 62:119-58(1982) and Dubowchik et al., 1999, Pharmacology and Therapeutics, 83,67-123.

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980.

An antibody with or without a therapeutic moiety conjugated to it can beused as a therapeutic that is administered alone or in combination withcytotoxic factor(s) and/or cytokine(s).

An antibody that is used solely for detection/quantification may beraised in other mammalian species e.g. rabbit and directed against ahomologue of human SC6, providing that the antibody cross-reacts andstill recognises human SC6 polypeptides for use in the invention (e.g.rabbit anti-rat taurine transporter (TAU 11) catalogue no. TAU-11S,Alpha Diagnostic International Inc, San Antonio, Tex. 78238 USA).

The invention will now be described with reference to the followingexamples, which should not in any way be construed as limiting the scopeof the present invention. The examples refer to the figures in which:

FIG. 1: shows the expression of SC6 mRNA in human dermal microvascularendothelial cells (HDMEC) under normoxic and hypoxic conditions over atime course 4-16 hours. Levels of SC6 mRNA were quantified by real timeRT-PCR. mRNA levels are expressed as the number of copies ng⁻¹ cDNA.

FIG. 2: shows the expression of SC6 mRNA in the breast cancer cell lines(MCF7 and MDA435) and in renal cell carcinoma cells (RCC4), undernormoxic (N) and hypoxic (H) conditions. The filled bars represent thehypoxic counts and the clear bars represent the normoxic counts. mRNAlevels are expressed as the number of copies ng⁻¹ cDNA.

FIG. 3: shows the tissue distribution of SC6 mRNA. Levels of SC6 mRNAwere quantified in a panel of normal tissues by real time RT-PCR. mRNAlevels are expressed as the number of copies ng⁻¹ cDNA.

FIG. 4: shows the expression of SC6 mRNA in seven matched breast tumourtissues. Tumour tissues are designated by (T) and matched normal tissuesare designated by (N). mRNA levels are expressed as the number of copiesng⁻¹ cDNA.

FIG. 5: shows the expression of SC6 mRNA in matched tumour and controltissue from the breast, cervix, colon, kidney, lung, thymus and uterus.Levels of SC6 mRNA were quantified by real time RT-PCR. mRNA levels areexpressed as the number of copies ng⁻¹ cDNA. The filled bars representthe tumour tissue counts and the clear bars represent the control tissuecounts.

FIG. 6: shows the expression of SC6 mRNA in various cancer cell lines.The panel of human cell lines investigated are as follows: humanosteosarcoma (MG63); breast carcinoma (MDA-MB453, MDA-MB468, BT20,BT474, CAL51, DU4475 and T47D); renal carcinoma (Wilm); transformednormal renal cell line (293); transformed normal fibroblast cell line(MRC-5); uterine sarcoma (MES-SA); cervical carcinoma (HeLa S3);pancreatic carcinoma (Mia Paca, Panc 1, PC3, PC3M); human dermalmicrovascular endothelial cells (HDMECQ and HDMECQ-VEGF). Levels of SC6mRNA were quantified by real time RT-PCR. mRNA levels are expressed asthe number of copies ng⁻¹ cDNA.

FIG. 7: shows the expression of SC6 mRNA in clinical and normallymphatic system samples, clinical: lymphoma samples 9-37 and normal:CD34+, per blood leukocyte, spleen and thymus. Additionally SC6 mRNAexpression is also shown for a panel of lymphoma/leukaemic cell lines,including, Burkitt's lymphoma (Raji, Daudi, CA46, Namalwa, Ramos); MUTUm, BL58, OZ, SUD HL6, KHM108, T-cell leukaemia (Jurkat); acute monocyticleukaemia (THP-1); chronic myelogenous leukaemia (K-562); acutemyelogenous leukaemia (KG-1); REC1 MV, K1106, K-231, AS293, BL115 andpromyelocytic leukaemia (HL-60). mRNA levels are expressed as the numberof copies ng⁻¹ cDNA.

EXAMPLE 1 Expression of SC6 mRNA Under Hypoxic Conditions

Growth of Primary Endothelial Cells Under Normoxic and HypoxicConditions

HDMEC cells were treated by exposing them to either normoxic or hypoxic(1% O₂) conditions for 4, 8, or 16 h (methodology for cell culture as inRatcliffe P et al., Nature, 1999, 399, 271-275). After exposure, thecells were harvested for mRNA preparation.

Gene Expression Microarray (GEM) Analysis

A detailed analysis of differential gene expression in HDMEC cells grownunder normoxic (control) or hypoxic (treated) conditions for 4, 8 and 16h was performed using the GEM™ microarray services offered by IncyteGenomics, Ltd, Paloaoto, USA). In brief, pairs of polyA+ mRNAs thatcorresponded to the key comparisons under investigation (in thisinstance hypoxia versus normoxia) were provided. Preparation of mRNA wasperformed using the Incyte protocol. Incyte, prepared labelled probesand conducted a competitive hybridization reaction with these probes onone of their standard human microarrays.

GEM analysis of primary endothelial cells showed that the gene for SC6was increased under hypoxia. TABLE 1 Fold change of SC6 from GEManalysis using 10,000 cDNA Incyte arrays Hypoxia (h) 4 8 16 Foldincrease in SC6 expression compared to 2.4 2.8 6.1 cells under normoxia

Quantification of SC6 mRNA by RT-PCR

To confirm the GEM analysis results, we used real time quantitativeRT-PCR (as described in Example 2) to analyse the expression of SC6 mRNAin primary endothelial cell cultures, HDMEC, cultured under normoxic andhypoxic conditions.

The expression of SC6 mRNA was quantified in HDMEC endothelial cellsafter 4, 8 and 16 h of exposure to normoxic or hypoxic conditions (FIG.1). In agreement with the results from GEM analysis, we found a dramaticincrease in SC6 mRNA levels after 16 h exposure to hypoxia relative tocells exposed to normoxic conditions.

The expression of SC6 mRNA was also quantified in a renal cell carcinomacell line, (RCC4), and in two breast carcinoma cell lines, (humancaucasian breast adenocarcinoma, ECACC, MCF7 and breast/mammary glandmetastatic site pleural effusion, ductal carcinoma, ATCC, MDA435),cultured under either hypoxic or normoxic conditions for 16 h (FIG. 2).

The presence of hypoxia was associated with a large increase in theexpression of SC6 mRNA in all cases indicating that SC6 expression isinduced by hypoxic factors.

EXAMPLE 2 Expression of SC6 mRNA in Human Tissues

Real time quantitative RT-PCR was used (Heid, C. A., Stevens, J., Livak,K. J. & Williams, P. M. Genome Res. 6, 986-994 (1996); Morrison, T. B.,Weis, J. J. & Wittwer, C. T. Biotechniques 24, 954-958 (1998)) toanalyse the distribution of SC6 mRNA in normal human tissues (FIG. 3)and in matched clinical normal and tumour cancer tissues (FIGS. 4 and5), and in tumour cell lines (FIG. 6).

Quantification of SC6 mRNA by RT-PCR

RT-PCR was used to quantitatively measure SC6 expression in normal humantissue mRNAs (Clontech), and in matched cancer tissues and normal tissue(Clontech). The primers used for PCR were as follows: sense 5′atcggctatgcctccgttgtaa 3′ (SEQ ID No.3) antisense 5′agttggtggagctgatggtgat 3′ (SEQ ID No.4)

Reactions containing 5 ng cDNA, prepared using Superscript first strandsynthesis for RT-PCR kit (Life Technologies), SYBR green sequencedetection reagents (PE Biosystems) and sense and antisense primers wereassayed on an ABI7700 sequence detection system (PE Biosystems). The PCRconditions were 1 cycle at 95° C. for 10 min followed by 40 cycles of95° C. for 30 sec and 65° C. for 1 min. The accumulation of PCR productwas measured in real time as the increase in SYBR green fluorescence,and the data were analysed using the Sequence Detector program vl.6.3(PE Biosystems). Standard curves relating initial template copy numberto fluorescence and amplification cycle were generated using theamplified PCR product as a template, and were used to calculate SC6 copynumber in each sample. The expression of SC6 mRNA is shown as a relativeexpression level of mRNA (copy number ng⁻¹ cDNA).

The expression of SC6 mRNA was low in most normal tissues, with thehighest levels of expression found in tonsil, trachea and normal mammarytissues (FIG. 3).

The expression of SC6 mRNA was examined in seven matched clinicalsamples of normal and breast tumour tissues (obtained with permissionfrom the IMM, Oxford, UK). In all cases, SC6 mRNA expression wasincreased in the tumour samples in comparison to the matched normaltissue sample (FIG. 4).

The level of SC6 mRNA was further quantified in matched normal andtumour tissue from breast, cervix, colon, kidney, lung, thymus anduterus samples (FIG. 5). These samples were obtained from BD BiosciencesClontech, 1020 East Meadow Circle, Palo Alto, Calif. 94303. We foundthat SC6 mRNA expression was increased in cervical (1/1), colon (2/3),kidney (1/1), lung (2/2), and uterine (1/1) cell carcinoma relative tothe corresponding matched control samples.

The expression of SC6 mRNA was also determined in a number of humantumour cell lines (FIG. 6). The following list of human cell lines wereinvestigated: human osteosarcoma (MG63); breast carcinoma (MDA-MB453;MDA-MB468; BT20, BT474, CAL51, DU4475 and T47D); renal carcinoma (Wilm);transformed normal renal cell line (293); transformed normal fibroblastcell line (MRC-5); uterine sarcoma (MES-SA); cervical carcinoma (HeLaS3); pancreatic carcinoma (Mia Paca, Panc 1, PC3, PC3M); human dermalmicrovascular endothelial cells (HDMECQ and HDMECQ-VEGF). These celllines were obtained from the following suppliers: American Type CultureCollection (ATCC), Manassas, USA; European Collection of Cell Cultures(ECACC), Salisbury, Wiltshire, UK and BioWhittaker House, Wokingham,Berkshire, UK and were grown in media according to the suppliersinstructions. We found that SC6 mRNA levels were high in certain breastcarcinoma cell lines and that SC6 mRNA levels were particularlyincreased in renal, uterine and pancreatic carcinoma cell lines.

Additionally, we examined SC6 mRNA levels in clinical and normallymphatic system samples. The pathologically validated clinical lymphomasamples were obtained from Ardais Corporation (Lexington, Mass., USA)following approval from the Institutional Review Board (FIG. 7).Additionally SC6 mRNA expression was compared in a range oflymphoma/leukaemic cell lines, including, Burkitt's lymphoma (Raji,Daudi, CA46, Namalwa, Ramos); T-cell leukaemia (Jurkat); acute monocyticleukaemia (THP-1); chronic myelogenous leukaemia (K-562); acutemyelogenous leukaemia (KG-1); promyelocytic leukaemia (HL-60)—allavailable from ATCC or ECACC— and other leukaemia cell lines, MUTU III,BL58, OZ, SUD HL6, KHM108, REC1 MV, K1106, K-231, AS293, and BL115. Asfor the carcinoma cell lines in FIG. 3, certain lymphoma/leukaemia cellslines showed increased SC6 mRNA expression, including, Burkitt'slymphoma (1/5), acute monocytic leukaemia (1/1), chronic myelogenousleukaemia (1/1) cell lines and SUD HL6.

Similarly an increase in SC6 mRNA was observed in 65% of the clinicallymphoma samples as compared with normal lymphatic system samples. Insuch instances SC6 would be a good target for therapeutic interventione.g. with a human anti-SC6 antibody or with agents that inhibit ordown-regulate the expression or activity of SC6.

In the clinical setting it is likely that certain regions of a cancerare hypoxic. Our findings indicate that SC6 is upregulated in responseto hypoxia. It may be the case that (for some or all of the cells) thecancerous tissue is deregulated with respect to its' hypoxic responseand thus, SC6 is constitutively over-expressed.

In summary, SC6 shows a low expression profile in normal tissues but iselevated in certain tumour tissues and cancerous cell lines e.g.cervical, colon, renal, lung, uterine, breast and pancreatic cellcarcinoma, in addition to, Burkitt's lymphoma and myeloid leukaemia. Inparticular, SC6 is elevated under hypoxic conditions indicating that itmay be a cellular marker of hypoxic tumours.

SC6 up-regulation may serve to increase taurine levels within thehypoxic tumour cell and hence protect the tumour cell from cell damagedue to low oxygen concentrations. Furthermore, this protection mechanismwould enable the continued proliferation and motility of the tumourcells. Thus, SC6 may have a use as a marker in the diagnosis of, or as atarget in the treatment of, cancer.

The present invention is not to be limited in terms of the particularembodiments described in this application, which are intended as singleillustrations of individual aspects of the invention. Functionallyequivalent methods and apparatus within the scope of the invention, inaddition to those enumerated herein, will be apparent to those skilledin the art from the foregoing description and accompanying drawings.Such modifications and variations are intended to fall within the scopeof the appended claims.

The contents of each reference, patent and patent application cited inthis application are hereby incorporated by reference in its entirety.Preferred features of each aspect of the invention are as for each ofthe other aspects mutatis mutandis.

1. A method of screening for and/or diagnosis of hypoxia relatedconditions in a subject and or monitoring the effectiveness of therapyfor said condition, which comprises the step of detecting and/orquantifying in a biological sample obtained from said subject an SC6polypeptide which a) comprises or consists of the amino acid sequence ofSEQ ID No. 1; b) is a variant having one or more amino acidsubstitutions, deletions, insertions or modifications relative to theamino acid sequence of SEQ ID No. 1 provided that such variant exhibitsthe immunological and/or transporter activity of the polypeptide withthe amino acid sequence of SEQ ID No. 1; or c) is a fragment of apolypeptide as defined in a) or b) above, which is at least ten aminoacids long.
 2. A method of screening for and/or diagnosis of hypoxiarelated conditions in a subject and or monitoring the effectiveness oftherapy for said condition, which comprises the step of detecting and/orquantifying in a biological sample obtained from said subject the amountof an isolated or recombinant DNA nucleic acid sequence which a)comprises or consists of the DNA sequence of SEQ ID No. 2, or its RNAequivalent; b) is a sequence which is complementary to the sequences ofa); c) is a sequence which codes for the same polypeptide as thesequences of a) or b); d) is a sequence which shows substantial identitywith any of those of a), b) and c); or e) is a sequence which codes fora variant or fragment of SEQ ID No.
 1. 3. An antibody that specificallybinds to an SC6 polypeptide as defined in claim
 1. 4. An antibodyaccording to claim 3 wherein the antibody is monoclonal, polyclonal,chimeric, humanised or bispecific, or is conjugated to a therapeuticmoiety, second antibody or a fragment thereof, a cytotoxic agent orcytokine.
 5. The method according to claim 1 wherein the polypeptide isdetected and/or quantified using an antibody that specifically binds toan SC6 polypeptide.
 6. A method of screening for agents that modulate(i) the expression or activity of an SC6 polypeptide as defined in claim1, or (ii) the expression of a nucleic acid molecule, said nucleic acidmolecule comprising an isolated or recombinant DNA nucleic acid sequencewhich a) comprises or consists of the DNA sequence of SEQ ID No. 2, orits RNA equivalent; b) is a sequence which is complementary to thesequences of a); c) is a sequence which codes for the same polypeptideas the sequences of a) or b); d) is a sequence which shows substantialidentity with any of those of a), b) and c); or e) is a sequence whichcodes for a variant or fragment of SEQ ID No. 1, said method comprisingcomparing the expression or activity of said polypeptide, or theexpression of said nucleic acid molecule, in the presence of a candidateagent with the expression or activity of said polypeptide, or theexpression of said nucleic acid molecule, in the absence of thecandidate agent or in the presence of a control agent; and determiningwhether the candidate agent causes the expression or activity of saidpolypeptide, or the expression of said nucleic acid molecule, to change.7. A method of screening for agents that interact with an SC6polypeptide, said method comprising contacting said polypeptide with acandidate agent and determining whether or not the candidate agentinteracts with said polypeptide.
 8. The method of claim 6 wherein theexpression or activity level of said polypeptide, or the expressionlevel of said nucleic acid molecule is compared with a predeterminedreference range.
 9. An agent identified by the method of claim 6, whichinhibits or down-regulates the expression or activity of saidpolypeptide, or the expression of said nucleic acid molecule.
 10. Amethod for the prophylaxis and/or treatment of a subject suffering froma hypoxia related condition, which comprises administering to saidsubject a therapeutically effective amount of a member selected from thegroup consisting of: (i) an SC6 polypeptide as defined in claim 1, (ii)a nucleic acid molecule, said nucleic acid molecule comprising anisolated or recombinant DNA nucleic acid sequence which a) comprises orconsists of the DNA sequence of SEQ ID No. 2, or its RNA equivalent; b)is a sequence which is complementary to the sequences of a); c) is asequence which codes for the same polypeptide as the sequences of a) orb); d) is a sequence which shows substantial identity with any of thoseof a), b) and c); or e) is a sequence which codes for a variant orfragment of SEQ ID No. 1, and (iii) an agent which inhibits ordown-regulates the expression or activity of an SC6 polypeptide. 11.(canceled)
 12. (canceled)
 13. The method according to claim 10, whereinthe agent is an antibody that specifically binds to an SC6 polypeptide.14. The method according to claim 1, wherein the hypoxia relatedcondition is selected from cancer, angiogenesis and angiogenesis relateddisorders.
 15. The method according to claim 14 wherein the cancer isselected from cervical, colon, renal, lung, uterine, breast orpancreatic cell carcinoma, lymphoma and leukaemia.
 16. The methodaccording to claim 2, wherein the hypoxia related condition is selectedfrom cancer, angiogenesis and angiogenesis related disorders.
 17. Themethod according to claim 5, wherein the hypoxia related condition isselected from cancer, angiogenesis and angiogenesis related disorders.18. The method according to claim 10, wherein the hypoxia relatedcondition is selected from cancer, angiogenesis and angiogenesis relateddisorders.
 19. The method according to claim 16 wherein the cancer isselected from cervical, colon, renal, lung, uterine, breast orpancreatic cell carcinoma, lymphoma and leukaemia.
 20. The methodaccording to claim 17 wherein the cancer is selected from cervical,colon, renal, lung, uterine, breast or pancreatic cell carcinoma,lymphoma and leukaemia.
 21. The method according to claim 18 wherein thecancer is selected from cervical, colon, renal, lung, uterine, breast orpancreatic cell carcinoma, lymphoma and leukaemia.